This research is directed toward the further elucidation of the enzyme mechanisms involved in the assimilatory reduction of nitrate to ammonium. The significance of this pathway is evident from the realization that the various oxidized inorganic forms of nitrogen represent, for all life forms, the ultimate nitrogen source. Nitrate assimilation is a two step process: the 2e reduction of nitrate to nitrate, followed by the 6e reduction of NO2 to ammonium. The enzymes catalyzing these reactions in Neurospora crassa, namely, nitrate reductase and nitrite reductase, are adaptively formed in the presence of either NO3 or NO2. Their synthesis is repressed by NH4 ion. NADPH-nitrate reductase, a sulfhydryl-containing molybdoflavoprotein possessing a functional b-type cytochrome, catalyzes the first step. The sequence of electron transfer mediated by this enzyme has been established: NADPH yields FAD yields Cytochrome b-577 yields Mo yields NO3. Knowledge of the nature of the enzyme catalyzing the second step, namely, nitrite reductase, is less complete. This laboratory has isolated preparations of significantly higher specific activity and stability than previously attainable and the enzyme has been characterized as a NAD(P)H-dependent, FAD-requiring metalloprotein. It stoichiometrically reduces NO2 to NH4 ion. The primary objectives at this point include the unambiguous elucidation of the electron transfer sequence which the nitrite reductase mediates by: (a) confirmation of the presence and function of "siroheme", a tetrahydroporphyrin moiety in the enzyme, through chemical, spectral and kinetic analysis, and (b) examination of the partial electron transport reactions which comprise the overall action of this enzyme by the use of artificial electron donors and acceptors, inhibitor studies and spectrophotometric observations of the enzyme during catalysis. Purification of the nitrite reductase (300,000 M.W.) to homogeneity, followed by determination of its subunit organization, will be undertaken. These results will be integrated with conclusions drawn from studies on the catalytic properties of the enzyme to gain insight into the relationships between the structure and function of the nitrite reductase.